Single molecule localization microscopy (SMLM) provided an unprecedented insight into the sub-nuclear organization of proteins and nucleic acids but apart from the nuclear envelope the role of the nuclear lipids in the functional organization of the cell nucleus was less studied. Nevertheless, nuclear lipids and specifically phosphatidylinositol phosphates (PIPs) play increasingly evident roles in gene expression. Therefore, here we provide the SMLM-based approach for the quantitative evaluation of the nuclear PIPs distribution while preserving the context of nuclear architecture. Specifically, on the example of phosphatidylinositol 4,5-bisphosphate (PIP2) we have:•Implemented and optimized the dual-color dSTORM imaging of nuclear PIP2.•Customized the Nearest Neighbor Distance analysis using ImageJ2 plug-in ThunderSTORM to quantitatively evaluate the spatial distribution of nuclear PIP2.•Developed an ImageJ2 tool for the visualization of the Nearest Neighbor Distance analysis results in cellulo.Our customization of the dual-color dSTORM imaging and quantitative analysis provide a tool that is independent of but complementary to the biochemical and lipidomic analyses of the nuclear PIPs. Contrary to the biochemical and lipidomic analyses, the advantage of our analysis is that it preserves the spatial context of the nuclear PIP distribution.

Biochemistry and Molecular Plant Biology Department Centro de Investigación Científica de Yucatán A C Calle 43 No 130 Colonia Chuburná de Hidalgo Yucatán Mérida C P 97200 Yucatán Mexico

Department of Biology of the Cell Nucleus Institute of Molecular Genetics of the Czech Academy of Sciences Prague Vídeňská 1083 142 20 Czech Republic

Faculty of Science Charles University Albertov 6 Prague 128 00 Czech Republic

Microscopy Centre of the Institute of Molecular Genetics of the Czech Academy of Sciences Prague Vídeňská 1083 142 20 Czech Republic

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